HMG CoA reductase inhibitors (statins) are known to prevent cardiovascular disease and improve lipid profiles. However, the effects of statins on renal outcomes, including decline in estimated glomerular filtration rate (eGFR) and proteinuria in patients with chronic kidney disease (CKD), are controversial. This meta-analysis evaluated the impact of statins on renal outcomes in patients with CKD.2) not requiring dialysis. The primary outcome was the differences in the change of eGFR. We also examined change of protein concentration in urine as a secondary outcome. A meta-analysis comparing statin and its control groups and a subgroup analysis examining intensity of statin were performed.We comprehensively searched the databases of MEDLINE, EMBASE, and Cochrane Databases. The inclusion criteria were published RCT and cohort studies comparing statin therapy to placebo or active controls in patients with CKD . In our subgroup analysis, those who received high-intensity statins had a significant difference in eGFR with a MD of 3.35 (95% CI: 0.91 to 5.79) ml/min/1.73 m2 compared to control. No significant change in eGFR was found with moderate- and low-intensity statin therapy. Compared with the control group, the statin group did not have a difference in reduction of proteinuria with MD in change of proteinuria of 0.19 gm/day (95% CI: -0.02 to 0.40).From 142 full-text articles, 10 studies were included in the meta-analysis. Overall, there was a significant difference in rate of eGFR change per year favoring statin group (mean difference (MD) = 0.10 ml/min/1.73 mOverall, there was a difference in change of eGFR between the statin and control group. High-intensity statins were found to improve a decline in eGFR in population with CKD not requiring dialysis compared with control, but moderate- and low-intensity statins were not. Statins were not found to decrease proteinuria in patients with CKD. Chronic kidney disease (CKD) is an important cause of death worldwide, affecting more than 10% of the population .There is evidence that statins may improve renal function and lower proteinuria in many prospective cohort studies, randomized-control trials and meta-analyses –5. This This systematic review and meta-analysis was conducted and reported according to established guidelines ,12 S1 A and was Two authors (AS and SU) independently searched published studies indexed in the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, MEDLINE, and EMBASE from January 1995 to January 2015. There were no limitations on language or publication date. References of selected retrieved articles were also examined. Sample search terms were: “statin”, “Hydroxymethylglutaryl-CoA Reductase Inhibitors”, “hmg coa reductase inhibitor”, “chronic renal insufficiency”, “kidney failure”, “CKD”. We limited searches to human only. We did not use filter for study design or limit for adults. Search terms that were used are detailed in We included all published randomized clinical trials (RCTs), prospective cohort and retrospective cohort studies comparing statins with placebo or no statin therapy for at least 6 months in patients with chronic kidney disease. We included cohort studies to explore renal outcomes and potential side effects from statin use. We excluded reviews, case reports, letters, commentaries, abstracts, and unpublished articles.2) and had a baseline eGFR, creatinine clearance or protein concentration in urine. Participants who received dialysis, renal replacement therapy or renal transplantation were excluded from the analysis.We included participants aged 18 years or older who had CKD stages 3 to 4  independently reviewed titles and abstracts of all citations that were identified. After all abstracts were reviewed, data comparisons between investigators were conducted to ensure completeness and reliability. The inclusion criteria were independently applied to all identified studies. Differing decisions were resolved by consensus.Full-text versions of potentially relevant papers identified in the initial screening were retrieved. If multiple articles from the same study were found, only the article with the longest follow-up period was included. Data concerning study design, participant characteristics, interventions, and outcome measures were independently extracted. We contacted the authors of the primary reports to request any unpublished data. If the authors did not reply, we used the available data for our analyses.A subjective assessment of methodological quality for RCT’s was conducted by two authors (AS and SU) on the following items, in which each component was categorized as having high, low or unclear risk of bias: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting. The quality of cohort studies was evaluated by the same authors using the Newcastle-Ottawa Scale (NOS) . The NOSWe performed meta-analysis of RCT and cohort studies separately. We reported the pooled mean difference (MD) of a total change in eGFR, proteinuria, rate of change in eGFR per year between statin group and controls. Pooled risk ratio (RR) of 50% reduction of eGFR and incidence of ERSD were also reported. For the purpose of our meta-analysis, creatinine clearance was considered to be equivalent to eGFR. The extracted studies were excluded from the analysis if they did not present an outcome in each of the intervention groups or did not have enough information required for continuous data comparison.We also performed a subgroup analysis of statin intensity, which was characterized as “high-intensity”, “moderate-intensity”, and “low-intensity” statin therapy, using definitions from the recent ACC/AHA guidelines (9). High intensity statins lower LDL-C by approximately ≥ 50%, include atorvastatin 40–80 mg and rosuvastatin 20-(40) mg. Moderate-intensity statins lower LDL-C by approximately 30% to <50%, include atorvastatin 10-(20) mg, rosuvastatin (5)-10 mg, simvastatin 20–40 mg, pravastatin 40-(80) mg, lovastatin 40 mg, fluvastatin XL 80 mg, fluvastatin 40 mg bid, pitavastatin 2–4 mg. Low-intensity statins lower LDL-C by approximately <30%, include fluvastatin 20–40 mg, lovastatin 20 mg, simvastatin 10 mg, pitavastatin 1 mg, pravastatin 10–20 mg. Statin and doses that are approved by the U.S. FDA but were not tested in the RCTs reviewed in the guidelines are listed in parentheses.2 statistic and Q statistic. For Q statistic, substantial heterogeneity was defined as P<0.1. The I2 statistic ranges in value from 0 to 100% . A sensitivity analysis was performed to assess the influence of the individual studies on the overall results by omitting one study at a time. Meta-regression was performed to find source of heterogeneity and to assess association between baseline LDL-C or percent reduction in LDL-C in the statin group and difference in eGFR change. Possible publication bias was assessed using funnel plot and Egger’s regression test [The heterogeneity of effect size estimates across these studies was quantified using the Iion test  . The MD of rate of eGFR change per year was 0.10 ml/min/1.73 m2 . The statistical between-study heterogeneity was absence . The MD of total change in eGFR was 1.78 ml/min/1.73 m2 (95% CI: -0.26 to 3.81). The statistical between-study heterogeneity was statistically significant with an I2 of 98% .We performed this meta-analysis to investigate the effects of statins versus placebo or control on eGFR and proteinuria using a random effects model. Since there is only one prospective cohort study , only RC. There was no significant change in urinary protein excretion with a MD of 0.19 gm/day (95% CI: -0.02 to 0.40). There was significant heterogeneity between the included studies in this meta-analysis (I² = 90%).A meta-analysis of studies examining change in proteinuria was also performed . There Only two studies were included in the analysis of comparison in incidence of ESRD and 50% reduction of eGFR or ESRD between statin and control groups. There were no significant association between statin group and control with ESRD , and 50% reduction of eGFR  .2 (95% CI: 0.91 to 5.79). No statistically significant difference of change in eGFR was found with moderate- (LDL-C reduction = 24%-32%) and low- (LDL-C reduction = 25%) intensity statin therapy. There were insufficient data to investigate effects of statins intensities on proteinuria.The results of our subgroup analysis of statin intensity is shown . There wTo assess the stability of the results of the meta-analysis of comparison in total change of eGFR and rate of eGFR change, sensitivity analyses were conducted by excluding one study at a time. None of the results was significantly altered, indicating that our results were robust.2 = 60%  on differences in total change of eGFR. Percent reduction in LDL-C was significant with a coefficient of -9.9 (95% CI: -18.2 to -1.5), p = 0.02, and R2 = 60% . In the To investigate potential publication bias, we examined the contour-enhanced funnel plot of the included studies in assessing total change in eGFR. Vertical axis represents study size (standard error) while horizontal axis represents effect size (log odds ratio). From this plot, bias is not present because there is symmetrical distribution of studies on both sides of the mean. The results of Egger’s test suggest that no evidence of publication bias was observed (P = 0.58). The funnel plot for assessing change in proteinuria was not performed due to too few studies.2 in the high-intensity statin group compared with control. However, we found no difference with moderate- and low-intensity statin therapy. In addition, we did not find that statins lowered proteinuria compared to the control group.Overall, our systematic review and meta-analysis in CKD participants found that high-intensity statin therapy ,21 had aOur findings are consistent with other meta-analysis in CKD participants not requiring dialysis ,26, whic2. It is possible that CKD patients had greater baseline renovascular disease and therefore required the greater effect of high intensity statins to provide a benefit on renal vasculature and to reduce greater oxidative stress. In addition, we demonstrated that LDL-C reduction had a significant association with change in eGFR. There is an increased change of eGFR with a greater reduction of LDL-C. This analysis helps to confirm a higher change of eGFR in high-intensity statins that had greater reduction of LDL-C compared with moderate- and low-intensity statins. Underlying mechanism of increased change of eGFR might be related to a higher reduction of oxidized LDL particles that normally cause renal and vascular damage. We also found an increased change of eGFR in higher baseline LDL-C. Higher baseline LDL-C may cause more renal injury than lower LDL-C. More severe renal injury may benefits from statin therapy more than lower injury [Previous meta-analyses of studies that reported renal function found that the effect of statins on eGFR may be dose-related in a general population . Howeverr injury ,28.Our findings are also unique in that we studied the effects of statin on hard end-points such as ESRD or 50% reduction of eGFR. We did not find a significant association of statins and these outcome. Our findings might be useful for clinicians to use high-intensity statin therapy to slow deterioration in renal function in CKD patients. However, there are no clear benefits of statin therapy on proteinuria. This might be because renal injury from lipotoxicity is independent of proteinuria .2 and a comprehensive MEDLINE, EMBASE and Cochrane Databases database search. Also, we included only studies with a treatment duration of at least 6 months, which allowed us to see the longer term effects of statins on renal function. In addition, we analyzed the difference in the change of renal function, and not only the end of treatment outcome as in a previous meta-analysis examining this topic [Strengths of our study include only enrolling CKD participants with a baseline eGFR less than 60 ml/min/1.73 mis topic . FurtherThere are several limitations in our meta-analysis, and thus our results should be interpreted with caution. First, we included a relatively small number of studies. This is because we separately analyzed studies with the outcome of total change in eGFR and rate of eGFR change per year as they used different units and we did not have enough information to change them into the same unit.Second, there is high heterogeneity between studies in the meta-analysis of statins effect on total change of eGFR. Potential sources of heterogeneity assessed by meta-regression were study designs, baseline LDL-C, and LDL-C reduction. We found that there was an increased in total change of eGFR with a higher baseline LDL-C or with an increased in reduction of LDL-C. This finding supports our main results of a significant effects of high-intensity statin.Our results add evidence that statin use in CKD patients may delay progression of kidney disease, and our results suggest that statins may have a dose-related effect on kidney function as only high-intensity statin significantly improved renal function assessed by estimated GFR. However, these results should be interpreted with caution. Further RCT’s using different statin intensities in CKD patients not requiring dialysis with a longer duration of study are needed.S1 Appendix(DOC)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S1 FigReview authors' judgments about each risk of bias. Item presented as percentages across all RCT and Non-Randomized trials. Positive signs represent low risk of bias. Negative signs represent high risk of bias. Blank spaces represent unclear risk.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigCircles represent each included study.(TIF)Click here for additional data file.


The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei. High-throughput imaging methods for brain-wide connectome mapping with precise location reference have been lacking. Here authors report a method that allows simultaneous acquisition of fluorescently labelled neurons and cytoarchitectural landmarks in the same mouse brain at the single-cell resolution. Mapping a cellular mouse connectome requires centimetre-scale imaging with axon resolution. Combined with physical sectioning, Li Here, we develop an automatic microscopy method called the brain-wide positioning system (BPS) for dissecting and locating neural structures with cytoarchitectonic landmarks at a single-cell resolution. BPS involves a whole-brain real-time counterstain protocol for simultaneously staining cytoarchitectonic landmarks during imaging and a high-throughput multi-channel brain-wide imaging system, termed wide-field large-volume tomography (WVT), for accelerating imaging acquisition at single-cell resolution. We obtain five full-volume, dual-colour mouse brain data sets of cell-type-specific fluorescent protein-expressing neurons and neuronal projections and subsequently reconstruct the three-dimensional (3D) fine structure of these neurons at the axon level using cellular landmarks. This standardized precision imaging approach facilitates quantitative brain-wide studies of 3D neuronal morphology, dendritic arbor and bouton distributions, axonal projection densities and distances and neuronal cell types, based on cytoarchitectonic landmarks at the cellular level. We propose that this method represents a routine tool for highly accurate and precise analysis of the cellular connectome.Thy1-GFP M-line transgenic mouseet al.As a proof of principle, we revealed the spatial distribution with anatomical annotation of an 8-week-old Thy1-GFP M-line transgenic mouse, including GFP-labelled neurons and propidium iodide (PI)-stained cytoarchitecture, was acquired at a voxel resolution of 0.32 × 0.32 × 1 μm in a coronal plane. The images of the typical structure of the hippocampus, cortex and cellular layers of the cortex (The whole-brain data set from a e cortex  demonstre cortex . The ovee cortex  illustrae cortex . Precisie cortex .Thy1-GFP M-line transgenic mouse at a voxel size of 0.32 × 0.32 × 2 μm in 77 h (n=14) and long-range (n=36) projection neurons according to the extent of the axonal arborizations. For long-range projection neurons, the axons extended toward the striatum or thalamus (n=16), midbrain (n=6) and pons or medulla (n=14), and most of these neurons had other collaterals that projected to distinct brain regions (n=24) and short-sparse (n=26) neurons , midbrain (n=5), and pons or medulla (n=3). Using these two data sets, we quantitatively compared the fine morphology of neurons projecting to the thalamus , n=21) with those projecting to the midbrain (midbrain group (MBG), n=11) , n=11) . The axo), n=11) , whereas), n=11) . The den), n=11)  and bran), n=11)  of the n), n=11)  and bran), n=11) . For the), n=11) , althoug), n=11) . These r), n=11) . FurtherThe co-localization of axonal long-range projections with cellular landmarks is a prerequisite for the precise analysis of the cellular connectome. To illustrate the accurate identification of the nuclei that the axons pass through and precise tracing of single axons, we performed two-channel whole-brain imaging at a voxel size of 0.32 × 0.32 × 2 μm on an 8-week-old C57BL/6J mouse injected with adeno-associated virus vector plasmid 12Notably, different from previous method for axon tracingSOM-Cre Cre ref.  mouse anCre ref.  and suggMoreover, we labelled brainstem neurons with long-range projections to olfactory bulb of a 7-week-old wild-type mouse. We imaged the entire brain using dual-colour channels through BPS at a voxel size of 0.32 × 0.32 × 2 μm . Sparse Here, we report a high-throughput method for the precise reconstruction and accurate localization of specific labelled neurons and projections in the whole brain. We observed the integrated cytoarchitecture of barrel columns in the barrel cortex using real-time counterstaining during whole-brain imaging and traced layer V/VI pyramidal neurons. In addition, we precisely traced a representative single-neuron projection on these PI-counterstained landmarks and identified the nuclei through which this projection passed. The diverse and complex spatial relationships between these neurons and nuclei suggest the necessity of reconstructing and analysing neuronal morphology and neural connections with accurate anatomical annotation. In this regard, BPS provides two major advantages.First, the acquisition of cytoarchitecture landmarks in whole-brain imaging is simple and feasible. In traditional neurobiological experiments, the registration of neural images to a planar brain reference atlas is typically required. Individual differences among subjects, unavoidable deformation during tissue preparation, and interval-sampling planar reference atlases inevitably generate location errors in these forced registrations. Thus, staining the cytoarchitecture landmarks and labelling the neural structures in the same brain are necessary to avoid these errors. However, low-permeability nucleic acid dyes, such as PI and DAPI, are traditionally used in slide staining to label cytoarchitecture landmarks, and these stains are difficult to use when staining the whole mouse brain. Here, we propose a new concept of real-time counterstaining during whole-brain imaging, rather than whole-brain counterstaining. The low permeability of nucleic acid dyes results in superficial staining during whole-brain imaging without background interference from deep layers in the same channel. Moreover, the sectioning-staining-imaging cycle guarantees consistent PI staining in all coronal planes. This real-time staining approach avoids additional sample preparation. The stained cytoarchitecture and labelled neurons in the same field of view (FOV) are simultaneously imaged. Thus, neither additional data acquisition time for anatomical landmarks nor extra two-channel registration is needed. This concept can be applied to other mechanical section-based whole-brain imaging techniques, such as serial two-photon (STP) tomography and fluorescence micro-optical sectioning tomography (fMOST), for acquiring anatomical references in the same brain.Second, WVT efficiently shortens the time required for data acquisition during full-volume whole-brain imaging at single-neuron resolution. The time required for data acquisition is one of the decisive factors for the use of whole-brain imaging as a routine approach in neuroanatomical studies. Currently, STP is considered the fastest approach; however, this approach sacrifices the continuity of the whole-brain imaging data set. fMOST and 2p-fMOST ref.  distinguRecently, the CUBIC techniqueCompared with current methods, BPS technology represents the technical advance of providing co-located cytoarchitecture for neural structure in the same whole brain. In addition, this technology also has the advantages of automation, high throughput, high resolution and robustness to accelerate the acquisition of high-resolution whole-brain data sets. BPS enables us to fast acquire both the neural structures and their own anatomic reference simultaneously. It helps to accurately identify and precisely annotate the brain-wide neural structures, which is difficult to be achieved by previous methods. Potentially, BPS can be routinely used to examine the intersubject variability of neurons. Consistent with previous studies202223et al.Full-volume imaging at high resolution inevitably generates large data sets, resulting in a crucial challenge to store, transfer, compute, manage and analyse these data. The most fundamental and essential challenge to mine large neural data sets is to recognize and reconstruct the complete morphology of single neurons from the raw data. This challenge is recognized worldwide as an open question and bottleneck. As a breakthrough, the BigNeuron project of the Allen Institute for Brain Science and the Blue Brain Project in Europe have focused on computational efforts. Recently, Markram In summary, the BPS method described in the present study facilitate the acquisition of a more detailed morphology of neurons and accurately identify the brain regions or nuclei in which these are located, connected with and passing through. We propose that this method has many potential applications for neuroscience and will promote future cellular connectome studies using a traditional interval-sampling 2D atlas to precisely generate ‘personalized' 3D maps for each brain at cellular resolution. This method could also contribute to cell type, projectome and connectome studies.Thy1-GFP M-line transgenic mice , a 12-week-old SOM-Cre transgenic mouse  and a 7-week-old wild-type mouse (mixed genetic strain) were used.All the animal experiments followed procedures approved by the Institutional Animal Ethics Committee of Huazhong University of Science and Technology or the Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University. An 8-week-old C57BL/6J male mouse, two 8-week-old −1) were prepared from 95% ethanol and 100% GMA.All histological procedures have been previously described317310f=150 mm) and a water immersion objective  and focused on the sample. The fluorescent light from the sample was collected through the objective and detected using two scientific complementary metal-oxide semiconductor cameras . The sensor array of the camera was 2,048 × 2,048 pixels with a 6.5 μm pixel size. A piezoelectric translational stage  moved the objective for axial scanning. The actual imaging format was set to 1,700 × 1,800 pixels to fit the size of the modulated light field of the DMD. The resin-embedded whole-brain sample was subsequently fixed using cyanoacrylate in a 200 × 90 × 53 mm sample box. The sample box was screwed onto a high-precision 3D translation stage . The 3D translation stage moved the sample for mosaic scanning and sectioning. The travel ranges were 200, 60 and 25 mm along the x, y and z axes, respectively. The encoded resolution of the x, y and z axes was 10, 1 and 4.5 nm, respectively. The repeatability of the x, y and z axes was ±200, ±75 and ±300 nm, respectively.The WVT system is shown in Sectioning and imaging were split in the WVT system, different from micro-optical sectioning tomography (MOST) and fMOST. For WVT, the microtome was used to only remove the imaged surface. The microtome was based on a 45° diamond knife , similar to MOST and fMOST.We controlled the 3D stage motion for sectioning and mosaic scanning using a computer. Running data acquisition and controlling other hardware in the WVT were achieved using customized C++ acquisition software in a workstation. The acquisition and motion control software were communicated via a TCP/IP protocol. The acquired data were saved as TIFF files in a storage array .−1 × 8 pixels), a 3D image stack of the beads was acquired with a z step of 200 nm. We reconstructed the x–z cross-section views of a selected bead in both channels, as shown in We imaged 0.2 μm FluoSpheres Carboxylate-Modified Microspheres  in the green and red channels to examine the point spread function of the WVT system. After setting the pattern period on the DMD plane to 109.44 μm  in the resin-embedded sample to obtain an optimized penetration protocol for the fluorescent nuclear staining solution. First, we studied the PI staining effect in GMA resin-embedded brain tissue at different concentrations and selected 2 μg mlntration  using th2CO3 into the water bath2CO3 during imaging.The sample was immersed in a water bath containing PI. Whole-brain imaging was performed in the water bath. To avoid the compromising effects of sectioning marks on the machined surface, the imaging plane was set slightly below the surface, generally at 1–2 μm. Before data acquisition, we focused on the top surface of the sample and adjusted the objective to obtain a clear image. Subsequently, we moved the objective down to set the imaging plane below the surface of the sample block. After these preparations, the imaging parameters were set, and the WVT system automatically performed the sectioning and imaging to complete the brain-wide data acquisition. We also flexibly adjusted the imaging parameters, such as range of interest, exposure time and so on. To enhance the in-focus GFP signal, we added Nax axis of the translation stage was the sectioning direction, and the sectioning width was 2 mm. The sectioning thickness was flexible. The y and z axes provided the necessary additional movement to cover the sample surface for sectioning. Thus, a smooth fresh surface was exposed for imaging.Sectioning was achieved through a relative motion between the fixed diamond knife and the 3D translation stage in the WVT. The π/2 were required to obtain an optical section image for each imaging channelSubsequently, the sample was imaged. PI solution served as the immersion liquid for the objective lens. The liquid level of the solution was maintained at a level higher than the bottom surface of the objective lens during data acquisition. The GFP and PI molecules were simultaneously excited, and the emitted fluorescence signals were divided using a dichroic mirror and detected using two cameras. Three phase-shifted raw images with a phase step of To reduce the data volume, the reconstruction of the SIM image was executed online using acquisition software. Only the reconstructed images were saved. The data set, comprising 1,700 × 1,800 pixel-sized mosaics, was saved at a 16-bit depth in the Lempel-Ziv-Welch (LZW) compression TIF format. After collection, the data set was sent to a PB-sized distributed storage via a standard 10-gigabit fibre.We acquired the data set in SOM-Cre mouse . 0.7 μl of CAV-Cre and 0.5 μl AAV-FLEx(loxP)-TVA-GFP were injected into the olfactory bulb  and the ipsilateral locus coeruleus  of a 7-week-old wild-type mouse, respectively. After surgery, the animals were returned to standard living conditions for 21 days until they were sacrificed for brain sample preparation.AAV-CAG and AAV-CAG-FLEX expressing GFP  were used as anterograde tracers. The stereotaxic coordinates for the target areas were based on the Mouse Brain in Stereotaxic Coordinates AtlasImage preprocessing for mosaic stitching  and illuWe visualized the data set using Amira software  to generate the figures and Movies. The preprocessed data set was imported into Amira software using a desktop graphical workstation . To process the TB-sized data on a single workstation, we transformed the data format from TIFF to the native LDA type using Amira. The visualization process included extracting the data in range of interest, sampling or interpolation, reslicing the images, identifying the maximum intensity projection, volume and surface rendering, and generating the Movies using the main module of Amira. The segmentation editor module of Amira was utilized for the manual outline segmentation of whole mouse brain.We applied the filament editor module of Amira to brain-wide tracing of long-range axons in 3D by human-machine interaction. From soma, we continuously loaded data blocks along axons and dendrites into Amira. We assigned initial and terminal points of fibres in the loaded block, and then Amira automatically calculated the path that the fibre walked along between these two points. This procedure was repeated until the whole neural morphology was reconstructed. The tracing results with original position information were saved in SWC format.We determined the barrel columns according to the counterstained images. The original PI-labelled data were sampled to 2 × 2 × 2 μm. According to the Mouse Brain in Stereotaxic Coordinates AtlasBesides, we used the NeuroGPSBecause recognition using the human eye is the golden standard of image segmenting in image processing323334t-test on two groups using SPSS software . The confidence level was set to 0.05 , and all results are presented as the means±s.e.m.We compared the lengths and branch numbers of the axons, apical dendrites and basal dendrites of neurons projecting to the thalamus (THG) and midbrain (MBG). All parameters were measured using Neurolucida Explorer . We performed Student's The authors declare that the data supporting the findings of this study are available within the article and its How to cite this article: Gong, H. et al. High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level. Nat. Commun. 7:12142 doi: 10.1038/ncomms12142 (2016).Supplementary Figures 1-9 and Supplementary Table 13D reconstruction of the GFP-labeled image block shown in Fig. 3 without preprocessing.The image dataset of the whole Thy1-GFP M-line mouse brain obtained via WVT. In each section, red represents a 2-μm coronal image of PI-stained cells, and green represents a 200-μm coronal image of the maximum intensity projection of GFP-labeled neurons.3.3D reconstruction of a pyramidal neuron and the surrounding barrel cortex area of the Thy1-GFP M-line mouse brain. The Movie shows a pyramidal neuron and its location from layer I to the corpus callosum . PI-stained cell bodies are in purple. The result shows that the soma of the pyramidal neuron is locating in layer V. The PI staining results clearly show layered structure of the barrel cortex and the precise locations of each cell body. The end of this Movie shows the reconstruction of the barrel walls. The size of data block is 760 × 850 × 150 μm2D consecutive slices showing the original images and axon tracing. The thickness of each slice is 16 μm, the size of display area is 220 × 160 μm, and the green signal is the tracing mark of the labeled axon on the current slice. The neuron is the green neuron in D5 in Fig. 4e.3 that the axon passed through at the voxel size of 0.32 × 0.32 × 2 μm. The tracing of the neuron is marked in red. There is 1-μm offset between tracing mark and volume data. Tracing the same pyramidal neuron as shown in Supplementary Movie 3 overlaid with the 3D image volume rendering.Tracing of a pyramidal neuron in the Thy1-GFP M-line mouse brain overlaid with 3D image volume rendering. The 3D image volume is rendered from 12 blocks of 320 × 320 × 300 μmProjections of pyramidal neurons in the barrel cortex of Thy1-GFP M-line brain. 3D view of Fig. 4d.3D reconstruction of the image dataset of the whole C57 mouse brain injected in Cg area with AAV-GFP and obtained via WVT. The same neuron as shown in Fig. 6. 3D volume rendering of the mouse brain is down-sampled at the voxel size of 2 × 2 × 2 μm due to the restriction of graphic card performance, and displayed with the manual outline segmentation of the whole mouse brain. Detailed volume views show the raw data in the thalamus, fibers in white and cell bodies in red. The size of the volume is 320 × 320 × 300 μm.3 , and the location of the data corresponds to the block in Supplementary Figure 6a. The red spheres were detected by NeuronGPS to show the centers of each soma.3D volume rendering of the injection site. The same brain as shown in Supplementary Movie 7. The data size is 540 × 660 × 180 μm2D consecutive slices showing the original images and AAV-labeled neuron tracing. The same neuron as shown in Fig. 6c.3D volume rendering of the AAV-labeled neuron. The same neuron as shown in Fig. 6c.


Single-chirality, single-wall carbon nanotubes are desired due to their inherent physical properties and performance characteristics. Here, we demonstrate a chromatographic separation method based on a newly discovered chirality-selective affinity between carbon nanotubes and a gel containing a mixture of the surfactants. In this system, two different selectivities are found: chiral-angle selectivity and diameter selectivity. Since the chirality of nanotubes is determined by the chiral angle and diameter, combining these independent selectivities leads to high-resolution single-chirality separation with milligram-scale throughput and high purity. Furthermore, we present efficient vascular imaging of mice using separated single-chirality  nanotubes. Due to efficient absorption and emission, blood vessels can be recognized even with the use of ∼100-fold lower injected dose than the reported value for pristine nanotubes. Thus, 1 day of separation provides material for up to 15,000 imaging experiments, which is acceptable for industrial use. Single-chirality, single-wall carbon nanotubes are attractive for bioimaging applications, however large-scale separation remains a challenge. Here, the authors utilize mixed surfactants for high-resolution single-chirality separation of  SWCNTs, demonstrating their improved performance for bioimaging. Therefore, the use of single-chirality SWCNTs was desired to improve efficiency. Of the chirality species defined by the chiral indices ,  SWCNTs have a well-designed electronic structure for biological imaging; S22 (724 nm)11 1The unique electronic and optical properties of single-chirality, single-wall carbon nanotubes (SWCNTs) have shown promise for developing next-generation of electronic, optoelectronic and biological applications124Various chirality separation methods have been developed to date, including density gradient ultracentrifugation (DGU)15171820In this study, we develop a new chirality separation method based on the newly discovered chirality selectivity between surfactant-wrapped SWCNTs and the gel. The selectivity is improved by the introduction of a mixed-surfactant system composed of SDS, sodium cholate (SC) and sodium deoxycholate (DOC). In this system, two different selectivities are found; chiral-angle selectivity in SC/SDS system and diameter selectivity in SC/SDS/DOC system. Since the chirality of SWCNTs is determined by the chiral angle and diameter, we demonstrate high-resolution single-chirality separation by combining these independent selectivities in the adsorption and desorption processes. High scalability of this method leads to industrial-scale separation of high-purity  SWCNTs, which provide improved performance in biological imaging.To clarify the effects of the mixed-surfactant system in gel chromatography, chirality selectivity between the surfactant-wrapped SWCNTs and the gel was investigated for two mixed-surfactant systems: SC/SDS and SC/SDS/DOC. HiPco SWCNTs (1.0±0.3 nm) were dispersed in an aqueous solution of 0.5% SC and 0.5% SDS. The SWCNT dispersion was loaded into a column filled with a dextran-based gel . After the elution of unbound metallic SWCNTs, adsorbed SWCNTs were eluted through a stepwise increase in surfactant concentration. For the SC/SDS system, the SDS concentration was increased in a stepwise manner with a fixed concentration of 0.5% SC. For the SC/SDS/DOC system, the DOC concentration was increased in a stepwise manner with fixed concentrations of 0.5% SC and 0.5% SDS. The chirality distribution of the eluted SWCNTs was characterized using optical absorption and photoluminescence spectroscopy.n,m) species) and their structural characteristics, such as diameter (D), chiral angle (θ) and smallest bond curvature radius  SWCNTs were selected as a target species for biological imaging.  SWCNTs have a similar trend to  SWCNTs in the SC/SDS/DOC system  and thus11, S22 and S33 peaks are clearly visible for each single-chirality SWCNT fraction, suggesting that high-purity single-chirality SWCNTs were obtained. Compared with those in For the small-scale separation , the resTo evaluate this method in comparison with others, the purities, production yields and throughputs of  and  were estimated from the optical absorption spectra. 1522 of  SWCNTs (725 nm)  SWCNTs, 0.52 mg ml−1 for s-SWCNTs and 0.64 mg ml−1 for p-SWCNTs. According to the absorption per unit mass estimated from the mass concentration,  SWCNTs exhibited enhanced excitation, which was 3.1- and 3.8-times higher than that of s-SWCNTs and p-SWCNTs, respectively.To use the separated  SWCNTs for biological imaging, the surfactant on the SWCNTs was replaced with a biocompatible surfactant, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene-glycol))-5000] (DSPE-PEG)(725 nm) . To make11 emission and/or suppressed S22 absorption, by a non-luminescent metallic species and/or a semiconducting species such as  and  that have longer photoluminescence wavelengths than 1,350 nm, where photons are absorbed by water. Further experiments will be necessary to understand this result more.To evaluate the relative fluorescence brightness, each sample was imaged under excitation at 735 nm . Despiten=4) were intravenously injected with the biocompatible  SWCNTs  and imaged under illumination by the 735-nm LED (n=2) injected with p-SWCNTs  were also imaged under the same imaging conditions. Nude mice  SWCNTs. 11 of  SWCNTs. This finding supports  SWCNTs as the best candidate for efficient imaging through skin. However, the wavelength dependence of the extinction properties differs depending on the biological tissue. In the case of imaging through a skull, it was reported that extinction minimum is in the 1,300 nm region due to contribution by scattering1328The advantage of 9,4) SWCNTs for biological imaging is not only the efficient absorption and photoluminescence intensity, but also their optimal photoluminescence wavelength for biological imaging. ,4 SWCNTsIn conclusion, we demonstrated high-throughput separation of high-purity, single-chirality SWCNTs using mixed-surfactant gel chromatography. The mixed-surfactant system composed of SDS, SC and DOC provided chirality selectivity between surfactant-wrapped SWCNTs and the gel: chiral-angle selectivity in the SC/SDS system and diameter selectivity in the SC/SDS/DOC system. By combining chiral-angle selectivity for adsorption and diameter selectivity for desorption, we developed a method for efficient single-chirality separation, resulting in the successful separation of 7 single species. This simple and fast process enabled the milligram-scale separation of single-chirality  and  SWCNTs with high purity (>90%), high yield and the high throughput of 5.2 mg per day. Since 100-fold scalability was confirmed in this method, we can apply it to larger systems, which may lead to enhanced throughput as needed. In addition, NIR fluorescence imaging of mouse vasculatures was performed using separated  SWCNTs as the fluorescence probe. Due to the high absorption and emission intensity per unit mass, the surfactant-exchanged, biocompatible  SWCNTs exhibited ∼20-fold brighter fluorescence than the pristine HiPco SWCNTs, providing clear vascular and deep inner-organ imaging with a high signal-to-noise ratio and high-temporal resolution. Blood vessels could be recognized even with the use of ∼100-fold lower injected dose than previously reported value for pristine SWCNTs, which help to reduce any possible risk for mice. The advantage of the minimum light loss through the skin and mucous tissues allow further advances in biological imaging. This efficient imaging can be performed 15,000 times at the maximum using the  SWCNTs from the output of one day of separation, which is acceptable for industrial use. This separation method has many advantages compared with the other reported methods because of its simplicity, low cost, high purity, high yield and high throughput. We believe that this industrial-scale single-chirality separation may be applied to the other single-chirality SWCNTs and could lead to new stages for various applications of SWCNTs.−2. To prevent heating during sonication, the bottle containing the sample solution was immersed in a bath of cold water. To remove the residual catalytic metal particles, nanotube bundles and impurities, the dispersed sample solution was centrifuged at 210,000g for 120 min in an angle rotor . The top 80% of the supernatant was collected and used for gel chromatography and biological imaging as pristine HiPco SWCNTs. Before separation, SDS , which is necessary for the separation of SWCNTsSWCNTs produced by high-pressure catalytic CO (HiPco) decomposition  with a content percentage of 5.2% were used as the starting material. First, 100 mg of HiPco SWCNTs was dispersed in 100 ml of a 0.5% SC  aqueous solution , which is known to be an efficient dispersant of SWCNTsApproximately 3 ml of allyl dextran-based gel beads  was packed in a plastic medical syringe equipped with a cotton filter on the outlet of the syringe. The adsorption procedure was performed as follows. After equilibration with a 0.5% SDS and 0.5% SC solution, 0.6 ml of the SWCNT dispersion with 0.5% SDS and 0.5% SC was loaded into the column. To elute unbound SWCNTs, the 0.5% SDS and 0.5% SC solution was loaded until no nanotubes were detected in the eluent. The elution procedures were different depending on the system. For the SC/SDS system, the adsorbed SWCNTs were eluted and collected by increasing the SDS concentration  with a fixed concentration of 0.5% SC. For the SC/SDS/DOC system, the adsorbed SWCNTs were eluted and collected by increasing the concentration of DOC  from 0.01 to 0.10% in 0.01% steps with fixed concentrations of 0.5% SC and 0.5% SDS. The chirality distributions of the eluted SWCNTs were determined using optical absorption and photoluminescence spectroscopy.−1. The adsorbed SWCNTs were eluted and collected by increasing the DOC concentration from 0.01 to 0.2% in 0.01% steps with fixed concentrations of 0.5% SC and 1.0% SDS. For the large-scale separation, ∼430 ml of Sephacryl S-200 gel was packed in a column . The adsorption procedure was performed in the same manner as for the small-scale separation. Approximately 80 ml of the SWCNT dispersion was loaded into the column at a flow rate of 10 ml min−1. After collecting fractions containing small-diameter SWCNTs at low DOC concentrations,  and  SWCNTs were eluted at 0.14 and 0.16% DOC, respectively. This procedure takes ∼6 h for a single cycle of separation. For further enhancement of the throughput, we changed the adsorption conditions and increased the amount of loaded SWCNT dispersion. The adsorption step was performed in a solution of 1.0% SDS, 0.5% SC and 0.13% DOC, and ∼400 ml of the SWCNT dispersion was loaded into the column.  and  SWCNTs were eluted at 0.14 and 0.16% DOC, respectively. This procedure takes ∼4.8 h for one cycle. The separation of other single-chirality SWCNTs , , ,  and ) is described in detail in the A chromatography system  was installed in a chamber with a defined temperature at 18–20°C. For the small-scale separation, ∼4.7 ml of allyl dextran-based gel beads (Sephacryl S-200 HR) was packed in a column . After equilibration with a 1.0% SDS and 0.5% SC solution, 1.0 ml of the SWCNT dispersion with 1.0% SDS and 0.5% SC, followed by the 1.0% SDS and 0.5% SC solution, was loaded into the column at a flow rate of 1 ml minThe optical absorption spectra of the SWCNTs were measured from 250 to 1,350 nm using a UV-vis-NIR spectrometer  with a quartz cuvette and a path length of 10 mm. The spectral slit width was 2 nm. The photoluminescence spectra of the SWCNTs were measured as a function of excitation and emission wavelengths using a spectrofluorometer  equipped with a liquid nitrogen-cooled InGaAs near-IR array detector. The excitation wavelength was varied from 450 to 850 nm in 5-nm steps, and the emission wavelength was varied from 850 to 1,350 nm. The spectral slit widths were 7 nm for both excitation and emission. The raw data were corrected using instrumental factors. The optical extinction spectrum of mouse skin was measured from 250 to 1,350 nm using a UV-VIS-NIR spectrometer  equipped with an integrating sphere. The mouse skin was completely removed from the back of a nude mouse and immediately used for the optical measurements. The thickness of the mouse skin was measured using digital calipers and estimated to be 0.35 mm. The spectral slit width was 8 nm.n,m) species. The optical absorption spectrum was fitted to the superposition of simulated peaks of the individual  species in the range from 800 to 1,350 nm, and the purity was computed as the ratio of the area of the dominant absorption peak to the sum of all peak areas. The production yield and the throughput were evaluated using the mass concentration, which was estimated from the absorbance in the ultraviolet region (∼280 nm). The absorption spectrum intensity was converted to the SWCNT mass concentrationThe purity was evaluated using a previously described methodg. The concentrate was diluted with water, and this process was repeated more than five times. Before use, the dispersion was centrifuged again for 90 min at 10,000g to remove any possible aggregates. For comparison, reference samples including semiconducting SWCNTs separated from HiPco SWCNTs and pristine HiPco SWCNTs were also prepared.The surfactant on the  SWCNTs was replaced with a biocompatible surfactant, DSPE-PEG (Sunbright)Female 6-week-old nude mice  were purchased from CLEA Japan, Inc., and housed for 8 weeks. All animal experiments were performed in accordance with the regulations approved by the Animal Care and Use Committee of AIST. For comparative vascular imaging, solutions of  and pristine HiPco SWCNTs dispersed by DSPE-PEG were intravenously injected into the tail vein of a mouse using a 29-gauge needle. Each mouse was anaesthetised by inhalation of isoflurane inhalation solution (Pfizer Inc.) and placed on the imaging stage within 5 min of injection. For dynamic imaging, a 29-gauge needle with a cannula  was inserted into the tail vein before anaesthetisation. The  SWCNT dispersion was injected to the anaesthetised mouse after the imaging began. For low-dose imaging, the  SWCNT dispersion was diluted with a 1% DSPE-PEG solution. All SWCNT dispersions were mixed with 10 × PBS to obtain a final PBS concentration of 1 × before injection. For each mouse, 100 or 200 μl of the SWCNT solution was injected.−2. Temperature raise of the mouse body by LED irradiation was confirmed to satisfy the regulations approved by the Animal Care and Use Committee of AIST. The exposure time for all images was 100 ms. The numerical aperture of the lens was adjusted depending on the experiments: 2.8 and 4 for comparative vascular imaging in 100 and 200 μl injection, respectively, 5.6 for dynamic imaging, and 1.4 for low-dose imaging.NIR fluorescence images were collected using a home-built imaging system . The excThe authors declare that the data supporting the findings of this study are available within the article and its How to cite this article: Yomogida, Y. et al. Industrial-scale separation of high-purity single-chirality single-wall carbon nanotubes for biological imaging. Nat. Commun. 7:12056 doi: 10.1038/ncomms12056 (2016).Supplementary Figures 1-12, Supplementary Methods and Supplementary References.Time-resolved NIR fluorescence images of a mouse injected with  SWCNTs.


In addition, we evaluated the phytochemical content in the extract and fractions of M. sylvestris and demonstrated that polyphenols were the most frequent group in all samples studied. An in vitro dual-chamber model to mimic the periodontal structure was developed using a monolayer of epithelial keratinocytes (OBA-9) and a subepithelial layer of fibroblasts (HGF-1). The invasive periodontopathogen A. actinomycetemcomitans (D7S-1) was applied to migrate through the cell layers and induce the synthesis of immune factors and cytokines in the host cells. In an attempt to analyze the antimicrobial properties of MSE and fractions, a susceptibility test was carried out. The extract  and chloroform fraction  were found to have inhibitory activity. The extract and all fractions were assessed using a cytotoxicity test and results showed that concentrations under 100 μg/mL did not significantly reduce cell viability compared to the control group . In order to analyze the inflammatory response, transcriptional factors and cytokines were quantified in the supernatant released from the cells. The chloroform fraction was the most effective in reducing the bacterial colonization (p< 0.05) and controlling inflammatory mediators, and promoted the down-regulation of genes including IL-1beta, IL-6, IL-10, CD14, PTGS, MMP-1 and FOS as well as the reduction of the IL-1beta, IL-6, IL-8 and GM-CSF protein levels (p< 0.05). Malva sylvestris and its chloroform fraction minimized the A. actinomycetemcomitans infection and inflammation processes in oral human cells by a putative pathway that involves important cytokines and receptors. Therefore, this natural product may be considered as a successful dual anti-inflammatory–antimicrobial candidate.The aim of this study was to investigate the  Aggregatibacter actinomycetemcomitans has been described as an important agent of localized aggressive periodontic lesions, but is also related to chronic periodontitis [A. actinomycetemcomitans and other pathogenic microbiota including Porphyromonas gingivalis, Treponema denticola, Tannerela forsythia trigger both innate and acquired immune responses, resulting in the progression of periodontal disease, and promote soft tissue inflammation and destruction with consequent bone resorption [Periodontal disease is characterized by bacterial infection associated with the presence of biofilm, resulting in chronic inflammation of the tooth-supporting tissues and leading to progressive destruction of periodontal tissue. This disease affects up to 90% of the world’s population , 2. Dentdontitis , 5. In asorption .The development of new therapeutic agents that can inhibit biofilm formation and modulate the inflammatory response will have a major impact on the prevention and treatment of periodontal disease .Malva sylvestris is one example. Commonly known as mallow, it is a plant native to Europe, North Africa and Asia. The ethnopharmacological literature has reported a long history of recognition for its potent anti-inflammatory, antioxidant, anticancer and antiulcerogenic properties [M. sylvestris contains phytochemicals including several classes of terpenoids, including monoterpenes, diterpenes, sesquiterpenes and norterpenes [in vitro the antimicrobial and anti-inflammatory activity of Malva sylvestris extract and fractions in a dual chamber model of epithelial and subepithelial cells infected by A. actinomycetemcomitans.Nature has been a source of medicinal products for centuries, yielding many useful drugs . A wide operties , 12. Somterpenes , 13, 14.terpenes . Thus, tMalva sylvestris leaves were purchased from a local farmer in the municipality of Princesa Isabel, Paraiba (northeast Brazil) in March and April 2013. This plant is not an endangered or protected species and was registered in the herbarium of the University of Sao Paulo (USP), receiving an identification number (ESA voucher # 121403). Absolute ethanol (800 mL) at room temperature was used to create extracts of M. sylvestris leaves (100 g) using exhaustive maceration (for 7 days). Filtration was used to obtain the ethanolic extract of M. sylvestris (MSE). The material was lyophilized, homogenized, weighed and stored at -20°C. The MSE was successively partitioned using liquid-liquid extraction with hexane, chloroform, and ethyl acetate solvents. The final residue obtained after ethyl acetate fractionation was totally soluble in water and thus was called the aqueous fraction (AF)[tion (AF). The extFor the flavonoid determination, the aluminum chloride method was used. Total flavonoid contents were calculated using quercetin for the calibration curve. The absorbance was measured at 425 nm with a microplate reader . The polyphenol content was measured by the Folin-Ciocalteu method and gallic acid was used as a standard equivalent . For theA. actinomycetemcomitans (D7S-1) was cultivated from the subgingival plaque of an African American female patient diagnosed with generalized aggressive periodontitis. The strain was kindly donated by Dr. Casey Chen  [Fusobacterium nucleatum ATCC 25586, Prevotella intermedia 25611 and Porphyromonas gingivalis ATCC BAA-308.The ifornia) . In addi2.Keratinocytes were processed and isolated, and the cell line established was named OBA-9 . The celA. actinomycetemcomitans DS7-1, Fusobacterium nucleatum ATCC 25586, Prevotella intermedia 25611 and Porphyromonas gingivalis ATCC BAA-308) to MSE extract and fractions were tested. Tests were performed according to Clinical and Laboratory Standards Institute guidelines [5 CFU/mL in 96-well microplates, using a trypticase soy broth and yeast extract medium  for A. actinomycetemcomitans and enriched with 5 μg/mL hemin and 1 μg/mL of menadione for the other microorganisms. The concentrations of MSE and fractions ranged from 3.125 to 1000 μg/mL. The vehicle control was ethanol , and the positive control was gentamicin . The plates for the evaluation of antimicrobial activity against facultative aerobes were incubated at 37°C, 5% CO2 and the plates for evaluation of activity against strict anaerobes were placed in an anaerobic chamber at 37°C, 10% H2, 10% CO2 and 80% N2. The MIC was defined as the lowest concentration of MSE or fraction that allowed no visible growth, confirmed by 0.01% resazurin dye . The minimum bactericidal concentration (MBC) was determined by subculturing in trypticase soy agar  or TSA containing 2 μg/mL hemin, 1 μg/mL menadione and sheep blood (5.0%) and 20 μL aliquots from each incubated well with a concentration equal to or greater than the MIC. The experiments were conducted in triplicate in three independent assays.The susceptibility of four potential periodontopathogenic bacteria (idelines . The min5 cells/mL) in a 96-well plate and incubated for 24 h at 37°C with 5% CO2. M. sylvestris extract and fractions (0.1–1000 μg/mL) were added to the cell culture and incubated for 24 h. After the incubation time, the supernatant was discarded and the cells were washed with PBS . Fresh medium and 20 μL of CellTiter-Blue  were added and incubated at 37°C and 5% CO2. The CellTiter-Blue test is a fluorescent assay that measures cell viability via non-specific redox enzyme activity. After incubation, the well contents were transferred to a new microplate and the fluorescence was read in a microplate reader  with 550 nm excitation, 585 nm emission [HGF-1 cells were seeded  and positioned in a 24 well plate. The basal chamber was seeded with HGF-1 fibroblasts. After 24 h the transepithelial resistance (TEER) was measured for each cell layer using a Millicell-ERS Volt-Ohm Meter . The cell layer confluence in the transwell insert was measured to reach the optimal TEER (>150 Ohm/cm2). On day 2, an overnight A. actinomycetemcomitans culture was harvested by centrifugation at 900 X g for 10 min at room temperature and incubated in the dual chamber with KSFM culture medium (~1x106 CFU/mL) passing through the upper layer of cells (OBA-9) and reaching the bottom layer (HGF-1) for 2h. Extracellular, unattached bacteria were removed by washing with saline buffer (PBS) two times. After this initial incubation, epithelial and subepithelial cell layers were incubated with gentamicin 100 μg/mL  to kill the extracellular bacteria. The medium was removed and washed with saline buffer. Fresh new culture medium was added and the culture was treated with MSE or fractions at a concentration of 75 μg/mL. In light of the dose-dependent effects of MSE and CLF treatments, this concentration was determined to be the highest concentration that possessed antimicrobial activity but was still non-cytotoxic after an exposure time of 24 h.The activity of MSE and its fractions in cells infected by iodontum . KeratinThe antimicrobial activity of MSE and fractions in the co-culture model was accessed after 24 h of treatment. Aliquots of 20 μL were cultured from each sample in TSB-YE plates to determine the CFU/mL and quantify the numbers of viable bacterial cells.2 First Strand Kit  according to the manufacture’s instructions. 84 genes were analyzed using inflammatory response & Autoimmunity Array RT2 profiler  with buffers supplied by the manufacturer. The full list of genes detected by the SYBR Green-optimized primer assays is shown in , according to the 2ΔΔCT method. DataSet is assigned a GEO accession number GSE72443.One microgram of RNA was converted in cDNA using RTshown in . A reactA. actinomycetemcomitans invasion in the lower chamber compartment upon reaching the subepithelial cells (HGF-1). In addition, we aimed to analyze genes related to the inflammation process to verify whether MSE and fractions could promote some biological activity in the infection process. RNA was isolated from cell culture after 24h of treatment using the RNeasy Mini Kit . Purity and quantity of RNA were measured in the NanoPhotometer P360 . RNA sample has been treated with DNase. Reverse transcription of RNA to cDNA was performed using the QuantiTect Reverse Transcription Kit  according to the manufacturer’s instructions. Based on PCR array analysis, genes were selected that presented significant levels of down-regulation . The threshold was manually adjusted within the logarithmic curve above the background level and below the plateau phase. A comparative Ct method was used to calculate the relative gene number. The relative gene copy number was calculated using the 2ΔΔCT method.Quantitative PCR (qPCR) was performed to evaluate the possible effects of the Cytokine assays were performed on all samples using specific enzyme-linked immunosorbent assay (ELISA) kits . The cytokines were selected in order to confirm the encoded genes that exhibited down-regulation in the gene expression analyses. The concentration of IL-1alpha, IL-1beta, IL6, IL8, IL10 and GM-CSF were measured according to the manufacturer’s instructions.The phytochemical characterization revealed that the total polyphenol contents of MSE, CLF and AF were 38%, 26% and 22% gallic acid equivalents, respectively. Tannin represented 0.02%, 0.54% and 0.8% catechin equivalent in MSE, CLF and AF respectively; flavonoid content was 2.7%, 7% and 5.6% quercetin equivalent in MSE, CLF and AF respectively.A. actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis. CLF was the most potent, with an MIC against A. actinomycetemcomitans of 150 μg/mL, an MIC against F. nucleatum of 500 μg/mL and an MIC against P. intermedia of 125 μg/mL. The MSE had the lowest MIC against P. gingivalis (15.6 μg/mL). AF had no inhibitory activity against any of the bacteria tested. Gentamicin was used as the positive control (10 μg/mL).The cytotoxicity of the extract and all fractions was assessed at concentrations of 0.1, 1, 10, 100 and 1000 μg/mL. AF did not affect the cell viability (p>0.05) at any of the concentrations tested when compared to the control group (non-treated). MSE and CLF reduced the number of viable cells at concentrations of 100 μg/mL and 1000 μg/mL (p<0.05). MSE and CLF were non-toxic at concentrations of 0.1, 1 and 10 μg/mL .A. actinomycetemcomitans infection. In addition, cells were analyzed for the ability to form an impervious epithelial layer by measuring the TEER. Dose-dependent antimicrobial effects were found for MSE and CLF treatment but high concentrations (≥ 100 μg/mL) were toxic for the cells; thus we tested the highest non-toxic concentration for all the samples. A sub-MIC concentration of 75 μg/mL was established. Results confirmed that treatment with 75 μg/mL MSE or CLF did not significantly affect the percentage of viable cells or the integrity of the tight epithelial conjunction . Twenty-four hours after the infection was initiated, the groups treated with MSE and AF had no ability to reduce or eliminate the invasion in the host cells; however, the number of microorganisms in the CLF group was reduced significantly compared to the control vehicle group (p<0.05)(In order to confirm the invasion assay of  (p<0.05).2 Profiler PCR Array and 84 genes were screened and analyzed using the SABiosciences web portal software. The transcriptional profile in the lower chamber cell lines after 24 hours invasion assay is showed on Alterations in mRNA transcript levels for all treatment groups were initially analyzed using the RTΔΔCT method was the non-treated control group. A minus-reverse transcriptase control was included in all qPCR experiments.The following genes were analyzed using qRT-PCR: IL-1alpha, IL-1beta, IL-6, IL-8, IL-10, CD14, PTGS, FOS, BCL6 and MMP-1. The reference control gene was GAPDH and the calibrator for the 2A. actinomycetemcomitans and treated with MSE had statistically significant down-regulation of the genes IL6, IL8, CD14 compared to the control group (non-infected cells) (p<0.05). The chloroform fraction down-regulated the gene expression of IL-1beta, IL6, IL10, PTGS, CD14, PTGS, FOS and BCL6 (p<0.05) compared to the control group. The group treated with AF showed a down-regulation of mRNA transcript levels for the genes IL-1alpha, IL-1beta, CD14, PTGS and FOS (p<0.05) (The results showed that cells invaded by (p<0.05) .The concentrations of IL-1 alpha, IL-1beta, IL-6, IL-8, IL-10 and GM-CSF were quantified by ELISA to confirm whether the proteins encoded by the down-regulated genes were found at reduced levels in the supernatant. MSE reduced the expression of IL-6 in the infected cells compared to the vehicle control group (p<0.05). Significant differences in the levels of a number of cytokines were found between the CLF and control groups. Specifically, reduced expression levels were observed for the cytokines IL-1beta, IL-6, IL-8 and GM-CSF . Infected cells expressed low levels of IL-1 alpha when treated with the AF, a decrease of 31% compared to the vehicle control group .Periodontal disease is an oral infectious inflammatory disease and the most common human chronic disorder . The relM. sylvestris [A. actinomycetemcomitans, a species known to be associated with periodontal disease, was used to model the infection of epithelial and subepithelial cell lines [M. sylvestris extract and fractions.For this study, we evaluated the toxicity, antimicrobial and anti-inflammatory activity of compounds naturally occurring in the plant lvestris . A screell lines . These rll lines . TranscrA. actinomycetemcomitans but also against other periodontopathogens  that are implicated in the development and virulence of periodontal disease [M. sylvestris works against both microaerophiles and anaerobes. In the literature, it has been shown that the ethanolic extract of M. sylvestris is effective as a bacteriostatic agent against methicillin-resistant S. aureus (I50 ≤ 32 μg/ml) [Helicobacter pylori (MIC ranged from 0.625 to >5.0 mg/mL)[Candida albicans [M. sylvestris have been reported in the literature for a few microorganisms [The antimicrobial susceptibility test showed that MSE and CLF had activity not only against  disease . In addi2 μg/ml) , and mod.0 mg/mL); moreoveus I50 ≤  μg/ml [Hrganisms , 31, 32 in vitro to provide better estimation of the potential of the compound as favorable therapeutic agent. The viability test showed that the CLF fraction was non-toxic at concentrations up to 100 μg/mL and AF had no toxic effects at any of the concentrations tested. The LD50 of the extract for cell lines OBA-9 and HGF  gave insight into the safe concentrations for use in the biological assays. M. sylvestris is widely known as a food or condiment and has been used for millennia in traditional medicine; however, only one in vivo test of its toxicity has been reported in the literature [Our findings also demonstrate that the bioguided fractionation was successful and may be a model for bioprospecting new drugs, as long as the active fraction (CLF) presented enhanced antimicrobial activity relative to the unfractionated extract. In addition, our data describe the cytotoxicity of the extract and fractions terature .A. actinomycetemcomitans affected the cell immune response and increased the production of local cytokines. All the treatments tested affected different signaling pathways. Upon treatment with the aqueous fraction, both the IL-1alpha gene and protein expression levels were reduced. The pro-inflammatory cytokine IL-1 and tumor necrosis factor alpha  are modulators of the host response to microbial infection. It has previously [The bacterial products from eviously  been demeviously .A statistical reduction of IL-6 gene expression and protein levels were found after treatment with the chloroform fraction (CLF). The higher expression levels of IL-6 in untreated periodontal disease might induce an increase in matrix metalloproteinases (MMPs) that are related to tissue destruction , 37. IL-In addition, the CLF treatment regulated the expression of other immunomodulatory genes , which indicates an effect on more than one signaling pathway and may result in a good therapeutic outcome. Finding compounds that trigger CD14 or toll-like receptors (TLRs) is potentially useful in periodontal disease. The binding of lipopolysaccharides (LPS) with CD14 might induce the temporary activation of many protein kinases and the phosphorylation of intracellular proteins essential for LPS activation in monocytes/macrophages .M. sylvestris may regulated the expression of cytokines in the inflammatory process. In a pre-clinical study, important anti-inflammatory action of the hydroalcoholic extract was found to interfere with the production of IL-1beta and consequently block leukocyte migration [M. sylvestris was found to have an immunomodulatory property, acting as a macrophage activators and promoting both IL-12 and (IFN) interferon transcripts [M. sylvestris in treating inflammation.The MSE could regulate the transcription of IL-8 but not the same cytokine expression. The answer to the question of how genomic information can be processed differently to produce a specific cellular proteome to date remains unanswered , 41. Theigration . Furthernscripts . OverallM. sylvestris showed a high occurrence of phenolic compounds in all studied extracts and fractions. This is consistent with a previous report [M. sylvestris. Furthermore, the interest in phenolic compounds has increased in recent years due to their possible implications for human heath, such as in treating and preventing cancer, cardiovascular disease and other pathologies [The phytochemical investigation of s report , in whichologies . OverallM. sylvestris may be considered as a potential drug candidate for use as a new therapeutic approach in the treatment of the periodontal disease.The results of the present study showed that the low-polarity fraction CLF has relevant dual activity, simultaneously controlling infection and inflammation processes. Thus, Malva sylvestris and its chloroform fraction were able to minimize the infection and inflammation process in oral human cells by a putative pathway that may involve the antimicrobial effect and modulation of cytokines and receptors. Therefore, this natural product may be considered as a successful dual anti-inflammatory–antimicrobial candidate.In our study we found that


Many studies have revealed shared music–language processing resources by finding an influence of music harmony manipulations on concurrent language processing. However, the nature of the shared resources has remained ambiguous. They have been argued to be syntax specific and thus due to shared syntactic integration resources. An alternative view regards them as related to general attention and, thus, not specific to syntax. The present experiments evaluated these accounts by investigating the influence of language on music. Participants were asked to provide closure judgements on harmonic sequences in order to assess the appropriateness of sequence endings. At the same time participants read syntactic garden-path sentences. Closure judgements revealed a change in harmonic processing as the result of reading a syntactically challenging word. We found no influence of an arithmetic control manipulation (experiment 1) or semantic garden-path sentences (experiment 2). Our results provide behavioural evidence for a specific influence of linguistic syntax processing on musical harmony judgements. A closer look reveals that the shared resources appear to be needed to hold a harmonic key online in some form of syntactic working memory or unification workspace related to the integration of chords and words. Overall, our results support the syntax specificity of shared music–language processing resources. Therefore, we will focus on this contrast. Overall, the arithmetic manipulation was more salient . This suggests that effects based on general task difficulty should be more easily visible in the arithmetic task. Thus, if there is an effect of language on music and it is due to general task difficulty, then the arithmetic control task should also show an effect.A pre-test . Closure judgements were required after each chord sequence. Participants were asked to answer a language/arithmetic comprehension prompt which followed one-third of the closure judgements (randomly chosen). Given the limited interest in the language/arithmetic task behaviour itself and in order to save time, we only included comprehension prompts on catch trials in order to ensure task adherence. Their unpredictable occurrence meant that the linguistic/arithmetic task had to be carried out throughout the experiment. Participants received feedback on their language/arithmetic accuracy every 20 trials.In the musico-linguistic part of the experiment participants completed 120 trials, 40 of which were critical trials containing a garden-path (ambiguous S-coordination) or non-garden-path sentence (NP-coordination) in the language task and a harmonic sequence with modulation in the music task. Critical linguistic items were randomly paired with critical musical sequences.Each language item was only shown in one version to each participant. The choice of item version was counterbalanced. Specifically, language item 1 might be presented in language condition A to participant 1, condition B to participant 2 and condition C to participant 3. For these three participants, the position of the item in the experiment was the same. Furthermore, the language item was paired with the same music stimulus for these three participants. For the next three participants, this item had a different position (determined by chance) and was matched with a different music stimulus (determined by chance). This way, a particular music–language item match could have no systematic influence on the results and the position in the experiment is counterbalanced for different language conditions. Each music item was presented once in every version to each participant. Trials were pseudo-randomized with the following constraints: (i) at least three trials between different versions of a music item, (ii) at most three filler or critical trials after each other, and (iii) at most three same answer conditions (prompt matching or not matching) after each other. The music stimuli were presented auditorily (608 ms per chord). The language stimuli were presented word by word (500 ms per word followed by 108 ms ISI) at the centre of the screen. The onset of a word presentation coincided with the onset of a chord.In the musico-arithmetic part of the experiment participants completed 80 trials, 40 of which were critical trials containing a hard or an easy arithmetic problem in the arithmetic task and a harmonic sequence with modulation in the music task. The other half were filler trials. The combination of musical and arithmetic items, counterbalancing and trial randomization was done in the same way as described for the musico-linguistic task, except that only 80 musical sequences were used  because of the reduced trial count. The arithmetic stimuli were presented in a similar way as the language material (500 ms per number or operator followed by 108 ms ISI).2.1.5typical closure rating behaviour. Instead, we based our analysis on the median. Specifically, we applied a subject-specific median-split analysis on ratings. For each participant, we coded ratings as either indicating high closure  or not. The analyses were performed on the proportion of trials in each condition which indicated high closure. This analysis strategy amplifies small differences in ratings between different concurrent task conditions while acknowledging that most critical trials are perceived as highly complete (high closure).The closure ratings of critical trials were negatively skewed with a median rating of 6 (of 7), i.e. our data displayed a ceiling effect likely because all critical sequences ended on an authentic cadence. Therefore, an analysis based on mean ratings appeared inappropriate as the mean would not be a good representation of the 2.22.2.1Mauthentic cadence=0.81>Mno cadence=0.12, difference s.d.=0.21). Therefore, all participants appear to have rated filler trial music as expected by music theory.Before presenting the crucial results of the critical trials, we first present evidence for good task adherence. Concerning the music task, using the filler trials which ended either in an authentic cadence (requiring high closure ratings) or not (requiring low closure ratings), we found that all participants had an equal or greater proportion of high-closure ratings for the authentic cadence endings than the no-cadence endings . As expected, the critical trial accuracy showed a clear difference between challenging ambiguous S-coordination  and less challenging NP-coordination trials =2.28, p=0.027, pη2=0.089). Arithmetic task accuracy was similar in general . As expected, the critical trial accuracy showed a clear difference between the hard  and the easy arithmetic trials =5.25, p<0.001, pη2=0.342).Language task accuracy  was high in general . Otherwise, first key endings were less likely  to receive a high closure rating than second-key endings . These two factors did not interact . Still, as shown in figure 3a, the simple main effects revealed a Difficulty effect only for first key endings , not for second key endings .The critical closure data of the musico-linguistic part of the experiment were analysed in a 2  × 2  within-subjects ANOVA; see figure 32.2.2.2b). There was no effect of arithmetical difficulty . Otherwise, first key endings were less likely  to receive a high closure rating than second-key endings . These two factors did not interact .The critical closure data of the musico-arithmetic part of the experiment were analysed in a 2  × 2  within-subjects ANOVA b. There 2.2.2.3F1,53=5.20, p=0.027, pη2=0.089). Otherwise, all three factors showed a main effect   . The Key and Task factors interacted  while Key and Difficulty did not . The three-way interaction was marginally significant .In order to see whether the difficulty effect in the musico-linguistic task was significantly different from the one in the musico-arithmetic task we performed a three-way within-subjects ANOVA on the harmonic closure ratings with the factors Task , Difficulty  and Key . Indeed, the difficulty effect was greater in the language task than in the arithmetic task  held in memory. This sixth word either linked two sentences (S-coordination) or two noun-phrases (NP-coordination) depending on the ninth word in the sentence. The arithmetic control task required no such retrieval of an item from memory. Therefore, it could be hypothesized that the different effects of linguistic and arithmetic difficulty are based on a working memory mechanism: one either has to retrieve a temporally distant element or not. Re-analysing a word held in memory because of an unexpected new word could be thought to impair the ability to simultaneously hold chords in working memory. This in turn might impair the ability to use them for harmonic integration. In order to evaluate this alternative scenario we included digit span as a covariate in the analysis of the musico-linguistic task data. If working memory, rather than common syntactic integration resources, was responsible for the language effect, then the greater availability of working memory resources should lead to a reduction in the influence of language on music. An ANCOVA analysis with z-scored digit span as a measure of working memory did not support this scenario. Neither forward digit span, nor backward digit span, nor overall digit span significantly modulated any of the main effects or interactions . This supports a functional distinction between syntactic processing and general working memory [However, a series of open questions remain concerning the effect of language on music ratings. Firstly, the difficulty in the language domain consisted in the syntactic re-analysis of a conjunction . The semantic manipulation consisted of semantic garden-path sentences  in which a disambiguating word leads to the semantic re-interpretation of an ambiguous word held in memory. Note that semantic garden-path effects have previously been shown to be modulated by a music harmony manipulation . This repost hoc nature of the analysis strategy we chose in experiment 1, i.e. the decision to focus on the proportion of high-closure ratings rather than raw ratings due to an unexpected ceiling effect. As opposed to the exploratory nature of experiment 1, experiment 2 was purely confirmatory and used the same analysis strategy.Finally, an additional reason for including a second experiment here is the 3.3.13.1.1Sixty-two participants were invited to take part in the experiment . None had participated in experiment 1. They were all Dutch native speakers, aged 21 on average (s.d.=3.0), with 3.3 years of musical training on average (s.d.=3.9). Forty-six were self-described non-musicians, 16 amateur musicians. They were paid € 12 or undergraduate course credits for their participation and were naive as to the purpose of the experiment.3.1.2We employed a 2 (Key: first key ending or second key ending) × 2  design for the language syntax part of the experiment and a 2 (Key: first key ending or second key ending) × 2  design for the semantic part of the experiment. All factors were manipulated within-subjects. As in experiment 1, auditory musical stimuli and visual linguistic stimuli were presented concurrently. The critical point in time was always the ninth position (underlined in examples 3 and 4 below), corresponding to the start of a new key in the musical material and the disambiguating word in the sentences.3.1.2.1We used the same music stimuli as in experiment 1.3.1.2.2hielp’/‘hielpen’, theson) in the object-extracted relative clause condition or the first noun phrase  in the subject-extracted relative clause condition.(3) Language example (syntactic manipulation)(3a) object-extracted relative clause (OR)The friendswhothe sonhelpedto get back on their feet let him see the building.Translation: (3b) subject-extracted relative clause (SR)The friendswhohelpedthe son get back on his feet let him see the building.Translation: As can be seen in example 3, 40 critical items were constructed in two versions each: an object-extracted relative clause and a subject-extracted relative clause. Sentences were 14 words long and were identical apart from the relative clause verb . Half the prompts required a ‘matches sentence’ response, half the opposite response.The 40 filler items included various syntactic constructions. Comprehension prompts of critical trials targeted the object- or subject-relative clause ambiguity  which was either ambiguous or not. The disambiguating word  was always the ninth word of the sentence.(4) Language example (semantic manipulation)(4a) semantic garden-path (GP)The programmer let hismouserun aroundon the table after he had fed it.Translation: (4b) non-garden-path (non-GP)The programmer let hisfield volerun aroundon the table after he had fed it.Translation: As can be seen in example 4, 40 critical items were constructed in two versions each: a semantic garden-path version and a non-garden-path version. Sentences were 14 words long and were identical apart from one manipulated word which occurred in positions two to seven (‘The mouse had been fed.—true). Half the prompts matched the sentence.Additionally, 40 filler items were included. They used various syntactic constructions and avoided semantic ambiguities. Comprehension prompts of critical trials targeted various parts of the sentence  participants were asked to complete sentence beginnings which did not include a disambiguating word . Sentence completions revealed that the intended word interpretation was overwhelmingly adopted in the non-garden-path condition  while this hardly happened in the semantic garden-path condition (13.3%). This is exactly the pattern expected of semantic garden-path sentences .In a first pre-test (3.1.4N=24) read sentences word by word while the timing of word presentation was under their control (self-paced reading). Afterwards they rated trials for difficulty. The semantic manipulation led to higher reading times from the disambiguating word onwards in the semantic garden-path condition (compared to non-garden-path condition). The difference was 44 ms (p=0.005) for the critical disambiguating word and 85 ms (p<0.001) for the post-critical word. Overall, this resulted in a greater perceived difficulty with these sentences (p=0.003). Moreover, the syntactic manipulation was equally salient as seen in reading times , on post-critical word: 58 ms (p=0.008)) and difficulty ratings . Thus, if we observe differences between the syntactic and the semantic manipulations these cannot be attributed to quantitatively different processing requirements of the two manipulations as they were matched in this respect.The aim of the second pre-test was to establish the strength of the difficulty manipulations of the syntactic and the semantic items. Participants (3.1.5The task was the same as in experiment 1. The type of manipulation (syntax or semantics) was blocked and the order counterbalanced. Experimental sessions did not include a working memory test. A testing session took approximately 90 min.3.1.6The analysis was the same as in experiment 1.3.23.2.1Mauthentic cadence=0.76>Mno cadence=0.09, difference s.d.=0.18).Before presenting the crucial critical trial data, we first show evidence of good task-adherence. Concerning the music task, using the filler trials we found, as expected, that all participants had a greater proportion of high-closure ratings for the authentic cadence endings than the no-cadence endings . As expected, prompts after object-relative clauses were answered less accurately  than those after subject-relative clauses . Accuracy during the semantics part was higher in general . There was no significant accuracy difference between garden-path  and non-garden-path trials . However, this null effect should not be taken to mean that the semantic garden-path effect was somehow absent. Instead, it reflects the fact that the comprehension prompts in the semantics part often did not target the semantic ambiguity of the sentences. As pre-test 2 showed, using the same comprehension prompts, critical semantics trials still exhibited a garden-path effect both in reading time and difficulty ratings, showing that it is not necessary to focus comprehension prompts on the garden-path manipulation in order for it to have an effect.Accuracy during the syntax part was high in general (3.2.23.2.2.1a). Object-relative clauses led to significantly fewer  high closure ratings than subject-relative clauses . Otherwise, first key endings were less likely  to receive a high closure rating than second-key endings . These two factors did not interact . Still, as shown in figure 4a, the simple main effects revealed a Difficulty effect only for first key endings , not for second key endings (t61<1).The critical closure data of the syntax part were analysed in a 2  × 2  within-subjects ANOVA a. Object3.2.2.2b). Closure ratings did not differ between semantic garden-path  and non-garden-path trials . Otherwise, first-key endings were less likely  to receive a high closure rating than second-key endings . These two factors did not interact .The critical closure data of the semantics part were analysed in a 2  × 2  within-subjects ANOVA b. Closur3.2.2.3F1,61=4.10, p=0.047, pη2=0.063). Otherwise, only one main effect was significant      .In order to see whether the influence of the syntax manipulation on music ratings was significantly different from the influence of the semantics manipulation we performed a three-way within-subjects ANOVA with the factors Manipulation , Difficulty  and Key . Indeed, the difficulty effect was different in the syntax part and the semantics part  was intermediate between the two syntactic manipulations . This makes a memory based explanation for the difference between the semantic and the syntactic effects unlikely.The difference between the syntactic effect and the semantic effect is remarkable. These two manipulations were rated as equally salient and led to very similar reading time changes. Nonetheless, the nature of the manipulation had different effects on the concurrent music perception. As discussed in §2.3, one could also propose a memory-based explanation for the difference between syntactic and semantic effects on music. Differences in the distance between the ambiguous word and the disambiguating ninth word could be responsible. However, when looking at these distances it becomes clear that the semantic manipulation  and establishing a new key (reflected in second-key ending ratings). The greater statistical power afforded by combining the data of the syntactic manipulations of both experiments might illuminate this issue.4.4.1F1,114=17.63, p<0.001, pη2=0.134) . The factor Experiment was without effect    . However, the greater power revealed a previously non-significant interaction between Difficulty and Key . Follow-up t-tests showed that the difficulty effect was specific to first-key-typical endings  while it was non-significant for second-key-typical endings . Note that including musical training as a covariate does not change the pattern of these results. In the overall ANCOVA z-scored musical training years did not modulate any of the main effects or interactions (ps>0.2); see the electronic supplementary material. In the electronic supplementary material, we also provide analyses by items.We combined the data of the syntax manipulations of experiment 1  and experiment 2  to form a single Difficulty factor . A 2  than in experiment 2 (pη2=0.094). Looking at the comprehension accuracy data reveals that this small difference might be related to participants sometimes giving up a full syntactic analysis of the very challenging object-relative clauses (accuracy=37%) but do so less in the challenging ambiguous S-coordination trials (accuracy=83%). This might indicate that syntactic integration resources can also be taxed too much, leading to incomplete parsing of the sentence [The comparison also showed that the syntax manipulation in experiment 1 appears to be numerically stronger  or harder (experiment 2) than their control task counterparts . Thus, in this study, there is no linear relationship between the effect of a task on music perception and its overall difficulty. General attention as well as shared syntactic resources might be involved in musical influences on language [We found no evidence for a general attentional mechanism driving the linguistic influence on music, as both control conditions were without effect. Thus, the nature of the manipulation (syntactic rather than something else) appears important. However, according to Perruchet & Poulin-Charronat’s  attentiolanguage ,11, whilet al. [Next to attention based explanations, an error based explanation of previous studies’ results ,4,6 was et al. . Howeveret al. ,5. Similet al. ,33). In et al.  as the let al. ,15,16, aet al. , or a wo6.This study found evidence that music and language share resources for syntactic integration . Our res